Cancer evades the immune system in part because a patient's T cells have been tolerized against potential antigens. Two immune-based strategies attempt to overcome this problem: one involves cancer peptide antigens used as vaccines and the other involves introduction of a cancer-antigen specific T cell receptor (TCR) into a patient's T cells, activated ex vivo (i.e. adoptive T cell therapies). The cross-reactivity of TCRs with self peptides that are structurally similar to the cancer peptide antigen will impact both of these approaches. We have recently conducted an in silico analysis of the human proteome to examine the number of structurally similar, HLA-A2-binding peptides, for each peptide among a collection of known cancer antigens. Our analysis revealed that that there was a 100-fold range of differences among cancer peptides in the number of such structurally similar peptides. Analysis of the mouse proteome showed that 40-60% of these structurally similar peptides were identical in both species. Based on these findings, we believe that we can use HLA-A2 transgenic mouse models to experimentally test two hypotheses that are directly relevant to these two immune-based cancer therapies. First, that the frequency of structurally similar self-peptides for a particular cancer antigen will influence the extent of T cell tolerance. Second, tha a higher frequency of structurally similar self-peptides in a proteome will increase the potential for off-target cross-reactivity in adoptive T cell approaches, especially with higher affinity TCRs The specific aims are: Aim 1. To determine the impact of the HLA-A2-peptide binding proteome on CD8+ T cell repertoires and tolerance. Aim 2. To determine the relationship between TCR affinity and peptide cross-reactivity. Aim 3. To perform a safety and efficacy assessment of TCR-transduced T cells in HLA-A2 transgenic mice.